Purified eukaryotic-initiation factor 4E having altered RNA binding affinity

ABSTRACT

The invention provides a method of purifying human eIF-4E protein and amino acid sequence variants thereof having altered binding affinity for capped RNA. Using the described purification, amino acid sequence variants can readily be expressed, purified and tested. Both lowered and enhanced binding affinity variants are useful for modifying protein expression levels in vivo and in vitro.

This application is a continuation of United States application Ser. No.08/995,060, filed Dec. 19, 1997, and now abandoned, which claims thebenefit of United States Provisional Application Ser. No. 60/033,533,filed Dec. 20, 1996.

ACKNOWLEDGEMENT OF GOVERNMENT FUNDING

Support for research leading to the invention was provided in part bythe National Institutes of Health, Grants GM40219 and CA63640-02. TheUnited States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION AND PRIOR ART

In eukaryotes, protein synthesis (translation) occurs in a complexprocess in which messenger RNA (mRNA) carrying amino acid sequenceinformation encoded in its nucleotide sequence interacts with ribosomesand a variety of cofactors and enzymes. Among the critical interactionsare those which occur in the initial steps of mRNA recognition duringinitiation of translation

Synthesis of mRNA occurs in the nucleus of the eukaryotic cell.Translation occurs in the cytoplasm. RNA sythesized in the nucleus issubject to modifications, generally termed processing reactions. Theseinclude capping, intron splicing and polyadenylation. Of importanceherein is the processing step known as capping. Capping is the addition,at the 5′ end of mRNA, of 7-methyl guanine, (m⁷G) joined by an unusual5′—5′ diphosphate bridge to the 5′ terminal ribonucleotide of mRNA. Thecapping reaction occurs naturally in the cell nucleus during mRNAsynthesis. Capping can also be carried out in vitro in anenzyme-catalyzed reaction. Commercially available kits can be obtained,for example, from Life Technologies, Inc., Gaithersburg, Md.

The initiation of translation in the cytoplasm requires specific bindingof proteins termed initiation factors. An important initiation factor inmammalian cells is the eukaryotic Initiation Factor—4E (eIF-4E) whichbinds to capped RNA (m⁷G-RNA). Translation is regulated in vivo byfactors and conditions which affect the binding of eIF-4E to m⁷G-RNA,including proteins that bind to eIF-4E (4E binding proteins). Forexample, at least one 4E binding protein designated 4E-BP-1 acts toprevent the binding of eIF-4E to m⁷G-RNA. 4E-BP-1, also known as PHAS-1,can undergo phosphorylation which is induced by insulin or other growthfactors. The insulin-induced phosphorylation of 4E-BP-1 releases thebound eIF-4E which is now available to bind m⁷G-RNA. This process mayaccount for the rapid stimulation of protein synthesis in muscle tissueinduced by insulin. Another eIF-4E binding protein is p220, also knownas eIF-4F, a protein that binds with eIF-4E as part of a functionalcomplex which interacts with mRNA to positively regulate translation.

The sequence of DNA encoding human eIF-4E has been determined [Reychlik,W. et al. (1987) Proc. Natl. Acad. USA 84: 945-949]. Yeast eIF-4E and afusion protein of mouse eIF-4E have been expressed in E. coli [Edery,I., et al. (1988) Gene 74:517-525; Edery, I., et al. (1995) Mol. Cell.Biol. 15: 3363-3371]. Haas, D. W. et al. (1991) Arch. Biochem. Biophys.284:84-89 reported purification of native eIF-4E from erythrocytes.Stern, B. D. et al. (1993) reported isolation of recombinant eIF-4Eusing denaturing concentrations of urea. However, expression andpurification of recombinant human eIF from the soluble fraction withouta denaturation step was not described before.

Transfection using RNA, has not been widely reported. The primarydifficulty is the susceptibility of RNA to RNAses and the lack of RNArestriction enzymes and ligases that has prevented in vitrorecombination of RNA segments. Nevertheless, transfection with RNA hasseveral advantages over transfection with DNA. Transfection by RNA doesnot normally lead to genetic alteration of host cells. Instead, atransient expression of the protein encoded by the transfecting RNA isobserved. There are circumstances where such transient expression ispreferable. For example, RNA transfected cells can transiently expressan antigen in an individual to be immunized. Garrity, R. R., et al.(1996) (Abstr. 1996 Meeting on Molecular Approaches to the Control ofInfectious Diseases, Cold Spring Harbor Laboratory, Cold Spring Harbor,N.Y., Sept. 9-13, 1996) reported that antibodies to gp 120 and gp 160 ofHIV-1 were detectable in guinea pigs that had been injectedintramuscularly with naked m⁷G-RNA encoding the respective antigens.Titres were low and the antibodies did not neutralize homologous virus.Since DNA transfection leads to chromosomal integration of extraneousDNA and long-lived expression of its encoded protein, unpredictable anddeleterious effects may occur in the host. Transient expressionresulting from RNA transfection can avoid these concerns. The problemsto be overcome with RNA transfections include extremely low transfectionefficiency and short intracellular lifetime of transfected RNA.

eIF-4E has recently been shown to play a direct role in maintaining thephenotype of breast cancer cells. The levels of eIF-4E in biopsies ofbreast cancer and breast cancer cell lines are increased (3-30 fold;mean of 10.5±0.9) as compared to benign fibroadenomas of breast tissueand control cells [Kerekatte, V. et al. (1995) Int J Cancer 64(1):27-31;Anthony, B. et al. (1996)Int J Cancer65:858-863]. Immunohistochemicalstudies showed that the cells expressing high levels of eIF-4E areindeed cancer cells and not stromal cells. In addition, evidenceindicates that high levels of expression of eIF-4E correlate with a poorclinical outcome in breast cancer [Li, B. D. L. et al. (1997) Cancer79(12):2385-2390]. A direct role for eIF-4E in breast cancer isevidenced by studies demonstrating that mammary carcinoma cells(MDA-435) exhibiting a 50% decrease in eIF-4E expression, due to stabletransformation with an antisense construct, have a markedly reducedability to produce tumors in nude mice. In addition, the down-regulationof eIF-4E expression in these cells results in relatively avasculartumors compared to control cells [Nathan, et al. 1997].

The cocrystal structure of mouse eIF-4E bound to m⁷GDP [Marcotrigiano J.et al. (1997) Cell 89:951-961] and the solution structure of yeasteIF-4E bound to m⁷GDP as determined by NMR spectroscopy [Matsuo H. etal. (1997) Nature Struct Biol.4:717-724] have been described. Bothstudies describe a cap-binding slot for eIF-4E in which the m⁷G moietyis sandwiched between the side chains of two tryptophans, Trp-56/Trp-102in mouse and Trp-58/Trp-104 in yeast eIF-4E. A third tryptophan, Trp-166(both mouse and yeast), as well as Glu-103 in mouse and Glu-105 inyeast, form hydrogen bonds with m⁷G. The cocrystal structuredemonstrated additional interactions involving residues Arg-157,Arg-112, and Lys-162 which make direct or water-mediated contacts withthe phosphate groups of m⁷GDP. The NMR solution structure of yeasteIF-4E showed that Arg-157, Lys-158 and Glu-159 are close to thephosphate tails of m⁷GDP and M⁷GTP

SUMMARY OF THE INVENTION

The invention provides purified recombinant human eIF-4E, as well as amethod of purification from transgenic cells expressing eIF-4E. Purifiedwild-type human eIF-4E binds in vitro to m⁷G-RNA with a binding constantof 10.1±0.3×10⁵M⁻¹. Binding is 1:1 on a molar basis, forming a binarycomplex designated eIF-4E-m⁷G-RNA. A sequence of amino acids involved inbinding human eIF-4E to m⁷G-RNA has been identified. Amino acidsubstitutions within the eIF-4E amino acid sequence have been made, someof which can result in 1-2 orders of magnitude tighter binding, othersof which result in reduced binding. The invention therefore alsoprovides modified human eIF-4E. The term “variant human eIF-4E” as usedherein embraces amino acid substitutions deletions and insertions andcombinations thereof affecting the binding affinity of the varianteIF-4E to m⁷G-RNA without destroying the protein's capacity to functionas an initiation factor.

The wild-type and variant human eIF-4E bound to m⁷G-RNA improvesstability of m⁷G-RNA, which enhances the transformation efficiency.Furthermore the presence of bound eIF-4E ensures immediate and efficienttranslation in the transfected host cell, which can be observed asenhanced expression of the protein encoded by the transfecting RNA. Theinvention therefore provides a method for making eIF-4E-m⁷G-RNA and amethod for transfecting eukaryotic cells by contacting the cells witheIF-4E-m⁷G-RNA. The method can be used with variant human eIF-4E orwild-type human eIF-4E. RNA transfected cells transiently express theprotein encoded by the RNA sequence.

The invention further provides a method for isolating 4E bindingproteins (4E-BP). Immobilized eIF-4E acts as an affinity ligand for thevarious proteins that bind to it and regulate translation. The 4E-BPproteins can thereby be isolated and characterized, in order to betterunderstand their role in controlling translation.

The invention further provides amino acid sequence variants of humaneIF-4E having either reduced or enhanced binding affinity for cappedmRNA (m⁷G-RNA). Variants having reduced binding affinity are useful intreatment of breast cancer. For example, DNA encoding a reduced-bindingvariant, introduced into breast cancer cells by a gene-therapy techniquecan act as a dominant negative mutant, counteracting the overexpressionof eIF-4E required to maintain the tumor phenotype of such cells.Variants having enhanced binding affinity have increased stability invitro and in vivo, for improved transient expression of a selected genein RNA transfection. Similar uses of natural or varied sequence eIF-4Efor temporally-limited gene regulation can be recognized by thoseskilled in the art, including, for example, to control thedifferentiation of stem cells. As a further utility, the ability of hostcells to express large proteins transgenically can be enhanced bytransfection using natural or variant eIF-4E.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Expression of eIF-4E in E. coli. A photograph is shown of aCoomassie Blue stained 10% polyacrylamide gel of whole cell lysates ofE. coli present in 0.5 ml samples of a culture immediately before, 2hand 3h after the addition of IPTG. The location of recombinant eIF-4E isdesignated by an arrow. The size of molecular mass markers present inthe first lane are indicated to their left.

FIG. 2. Purification of functional recombinant eIF-4E. A photograph isshown of a Coomassie blue stained 12% polyacrylamide gel of proteinspresent in starting material (E. coli lysate) (lane 1, 20 μg), m⁷GTPSepharose affinity purified eIF-4E (lane 2, 20 μg) and mono Q FPLCpurified (lane 3, 25 μg).

FIG. 3. Isoelectric focusing analysis of recombinant eIF-4E. Arepresentative analysis of recombinant eIF-4E (lane 2) and eIF-4Eisolated from rabbit reticulocyte lysate (lane 1) is shown. An arrowindicates the location of recombinant eIF-4E. (A) designates the regionwhere unphosphorylated and (B) the region where phosphorylated eIF-4Econcentrate (Bu et al. [1992] FEBS Lett. 301:15-18; Mick et al. [1998]FEBS Lett. 236:484-488).

FIGS. 4A-4B. Equilibrium binding constant for recombinant eIF-4E andm⁷GTP. The fluorescence intensity of recombinant human eIF-4E binding tom⁷GTP as a function of m⁷GTP concentration was measured. All solutionswere prepared in buffer A at pH 7.6, 25° C. and the concentration ofeIF-4E was 1 μM. An excitation wave length of 280 nm was used to monitorthe fluorescence intensity at 330 nm. Inset: Eadie-Hofstee plot of thefluorescence changes (ΔF), used to calculate the equilibrium bindingconstant for the eIF-4E/m⁷GTP complex formation.

FIG. 5. Production of immunoprecipitating anti-eIF-4E rabbit serum usingrecombinant eIF-4E. ³⁵S-labeled eIF-4E (arrow) prepared in reticulocytelysates was used in immunoprecipitation assays with preimmune (PI) orimmune (I) rabbit serum from two rabbits (Rb 1 & 2) (lanes 1-4). Anautoradiogram of the samples analyzed by SDS-PAGE is shown. Lane 6represents the quantity of [³⁵S]eIF-4E present in each incubation andlane 5 is a blank lane.

FIGS. 6A-6B. Isolation of eIF-4E binding proteins. Mammary carcinoma(184A) and Hep G2 cells were labeled with [³⁵S]methionine/cysteine.Equal aliquots of cell lysates were mixed at 4° C. with Protein ASepharose prebound with rabbit preimmune (lane 1) or anti-eIF-4E serum(lane 2), recombinant eIF-4E agarose beads (lane 3), and m⁷GTP Sepharose(lane 4). After washing, proteins bound to beads were analyzed bySDS-PAGE and autoradiography.

DETAILED DESCRIPTION OF THE INVENTION

Little is known about the molecular details of eIF-4E interactions withm⁷G-RNA or with regulatory proteins such as the 4E-BP proteins or withproteins of the eIF-4F complex. In order to characterize theseinteractions, the components thereof and their functions, sufficientquantities of purified eIF-4E must be available to the art. The presentinvention employs a transgenic expression system to synthesize humaneIF-4E and further provides a method for purification that now providessufficient quantities of human eIF-4E, to conduct physical and chemicalstudies. It now becomes possible to exploit the binding affinity ofeIF-4E for capped RNA to devise a novel RNA transfection vehicle.

RNA transfection has been limited heretofore by the extreme lability ofnaked RNA, by the lack of practical techniques for making in vitrorecombinant RNA constructs, and by a lack of transfection vectors.Nevertheless, RNA transfection has unique advantages for bio-medicalapplications. Whenever the goal is simply to express a gene product fora limited time, i.e., transiently, RNA transfection would be preferred.One such use, for example, is for the in vivo generation of antigenswhich, eliciting an immune response, serves as a means of generatingimmunity. The feasibility of such an approach has been demonstrated(Garrity, R. R., et al., supra). The present invention employs cappedRNA bound to eIF-4E (eIF-4E-m⁷G-RNA) as a transfection vector. Use ofeIF-4E-m⁷GRNA, improves RNA transfection efficiency and yield in twoways; by stabilizing the transfecting RNA and by assuring expression ofthe RNA once it has entered the host cell. Improved stability isobtained by the protective effect of eIF-4E. Once the eIF-4E-m⁷GRNA hasentered a host cell it can be translated immediately, without having tocompete with endogenous mRNA for endogenous initiation factor. AlthoughRNA transfection in animals has been demonstrated to occur simply byinjection of “naked” capped RNA, other carriers or complexing agents areuseful in nucleic acid transfections generally, as is well known in theart. Some examples include cationic lipid compounds, such asLipofectaminem™ (Life Technologies, Gaithersburg, Md.) and polycationicdendrimers, such as Starburst™ Dendrimers (Life Technologies,Gaithersburg, Md.).

Isolation of the 4E-binding proteins has been achieved using an affinitycolumn of eIF-4E bound to a chomatographic medium. Among the proteinsidentified as binding to such a column were p220 and eIF-4A, 220 kDa and48 kDa respectively [Sorenberg, N. et al. (1978) Proc. Natl. Acad. Sci.USA 75:4843-4847; Takara, S. M., et al. (1981) J. Biol. Chem.256:7691-7694). The use of eIF-4E affinity can isolate other 4E-bindingproteins as well. Other affinity strategies, for example, the use ofeIF-4E-m⁷G-RNA as an affinity capture ligand, can yield additionalproteins that function in translation.

The invention includes the demonstration that human eIF-4E cDNA can beexpressed in a prokaryotic host and purified as a soluble, activeprotein (termed recombinant human eIF-4E herein) from a lysate of a hostcell culture. As expressed in E. coli, eIF-4E is not phosphorylated. Thenon-phosphorylated protein is active and has biological and physicalcharacteristics that are similar or identical to native eIF-4E. In vitrophosphorylation of eIF-4E has a modest effect on its interaction withcapped mRNA. However, phosphorylation of eIF-4E appears to affectregulation of its interaction with regulatory proteins, for example, thep220 subunit of the eIF-4F complex.

The eIF-4E protein can be purified from host cells transfected toexpress the protein, as one aspect of the invention. The host cells canbe prokaryotic or eukaryotic, the choice of host being dependent onyield, growth characteristics, availability of suitable vector systems,and the like. The same can be said of the choice of expression vectorand the type of regulatory system used. For example, if desired,expression can be controlled in an inducible manner, to control thetiming of expression during the cell culture process. Further variationavailable to the skilled artisan is the choice of accumulating theexpressed protein within the cell or of causing its export into the cellculture medium. The purification method described herein is applicableto all such variants of host cell, regulation system and locus ofexpressed protein. The purification is exemplified with E. coli as hostcells, transformed with a T7 polymerase driven expression vector inwhich expression of the inserted gene is inducible. The eIF-4E proteinexpressed in these conditions was located primarily within the hostcells, rather than in the medium. A cell lysis step is thereforenecessary. At least a portion of the eIF-4E protein can be obtained insoluble form from a host cell lysate without resorting to denaturingconditions. Subsequent purification of soluble eIF-4E can be obtained bychromatography, using affinity separation and chromatofocussing ionexchange chromatography. In the exemplified purification, affinityseparation was carried out using m⁷GTP Sepharose. Chromatofocussing ionexchange employed the FPLC system (fast protein liquid chromatography)with Mono Q (Pharmacia AB, Uppsala, Sweden) ion exchange resin. Thepurification yielded a single detectable band on SDS-PAGE, and a singlecomponent by isoelectric focussing.

Purified human eIF-4E binds to m⁷GTP with an equilibrium bindingconstant of 10.1±0.3×10⁵M⁻¹ at pH 7.6 (20 mM HEPES buffer+1 mM DTT), 25°C. The result is similar (1.5-2 times higher) to results obtained usingnative human eIF-4E [Carberry, S. E. (1989) Biochemistry 28:8078-8083].Therefore purified recombinant human eIF-4E readily and spontaneouslybinds m⁷G-RNA under physiological conditions, in vitro. Single ormultiple amino acid substitutions as taught herein can result invariants of eIF-4E having higher binding affinities of at least 4 timesgreater than naturally-occurring eIF-4E. Other single or multiple aminoacid substitutions can result in decreased binding affinities form⁷G-RNA, without loss of other functions essential for initiatingprotein synthesis. Other sequence modifications, such as insertion ordeletion of one or more amino acids can be employed, alone or incombination with amino acid substitution, to modify eIF-4E bindingaffinity for m⁷G-RNA. The availability of milligram quantities of humaneIF-4E or modified eIF-4E makes it possible to make enougheIF-4E-m⁷G-RNA to render RNA transformation a practical reality. Inprinciple, capped RNA (m⁷G-RNA) encoding the desired amino acid sequencecan be prepared from host cells expressing the desired amino acidsequence, or by a combination of in vitro transcription and cappingreactions. In vitro transcription and capping reaction kits arecommercially available, for example from Ambion, Austin, Tex. BindingeIF-4E to m⁷G-RNA stabilizes the RNA, and increases the intracellularefficiency of translation, thereby enhancing the overall efficiency ofRNA transfection. Various transfection methods are available to thoseskilled in the art and are applicable to transfection by eIF-4E-m⁷G-RNA,of both cells in culture and cells in organized tissues and wholeorganisms.

Affinity chromatography provides a means for isolating proteins thatbind eIF-4E. Purified eIF-4E can be cross-linked to a chromatographicmatrix, for example agarose beads, and used as an affinity reagent. Thetechnique has yielded several proteins from cell lysates that havecharacteristics of subunits of the eIF-4F complex. In particular, theisolation of p220 protein by eIF-4E affinity chromatography appearspreferable to use of m⁷GTP sepharose or the use of anti eIF-4Eantibodies. Other proteins of interest that can also be isolated areeIF-4A, which is a subunit of eIF-4F, the 4E-BPs and other as yetuncharacterized regulatory proteins that are recruited to the 5′ cap oftranslationally active mRNAs.

Having pure recombinant human or modified eIF-4E at hand makes itpossible to generate polyclonal or monoclonal antibodies to eIF-4E. Theantibodies are of value for detecting and measuring eIF-4E, for exampleon gels, in solution or in vivo. Assay methods can be employed whereinthe analyte, eIF-4E, is labeled, for example with a radioactiveprecursor, or where the antibody itself is labeled, all according tomethodology known in the art. Polyclonal antibodies to purifiedrecombinant human or modified eIF-4E can immunoprecipitate bothphosphorylated and non-phosphorylated forms of eIF-4E. Anti-eIF-4E canbe used as an affinity reagent to provide an alternative affinityreagent for eIF-4E purification.

Studies on the binding site of eIF-4E have defined a sequence of aminoacids, involved in binding m⁷G-RNA extending approximately frompositions 112-123, numbering from the translation start site. The regionof amino acids involved in the binding of eIF-4E to m⁷G-RNA wasidentified using photoaffinity labeling with a labeled analog, [γ-³²P]8-N₃GTP. [Jayaram, B. et al. (1994) J. Biol. Chem. 269:3233-3242;Shoemaker, M. T. et al. (1993) J. Biol. Chem. 32:1883-1890; Salvucci, M.E. et al. (1992) Biochemistry 31:4479-4487]. The analog was found tobind to eIF-4E in competition with m⁷GTP and with capped RNA.Furthermore, the binding site was saturated by the analog, indicatingclose approximation of the analog to the normal binding site. The analogbinding to eIF-4E was rendered irreversible by a photo-chemical reactionand a tryptic peptide bearing the analog label was isolated byaluminum-III chelate chromatography and reversed phase HPLC. Once thebinding region peptide was identified, it was then possible to employalanine-scanning mutations to determine the contribution of specificamino acid residues to the binding, and to generate modified eIF-4Eproteins with altered binding affinities. Modified eIF-4E analogproteins having either increased or decreased binding affinities havebeen made. For example, substitution of alanine for the naturallyocurring tryptophane at position 113 (W 113 A) reduces affinity, whilesubstitution of alanine or a polar but uncharged amino acid for apositively charged amino acid enhances binding affinity. Enhancedbinding affinity can lead to increased capped RNA stability andincreased duration of expression. Decreased binding affinity can be ofvalue where sensitivity to regulatory influences is desired. Once thebinding site has been identified it is straightforward to generate aminoacid substitutions that have the desired binding characteristics withrespect to m⁷G-RNA. Accordingly, variant recombinant human eIF-4E is apart of the present invention.

Despite substantial progress in modeling the eIF-4E molecule in itsnative confirmation, the location of specific amino acid residues whosealteration affects binding affinity has remained unpredictable. Aminoacid loci identified herein as significanlty affecting m⁷G-RNA bindingdiffers from those previously identified on the basis of the cocrystalstructure [Marcotrigiano, J. et al. (1997) Cell 89:951-961]. Furtherelucidation of critical loci can be obtained by site directedmutagenesis or by random mutagenesis, given the herein-enabled methodsfor expression, synthesis and purification of eIF-4E.

eIF-4E/m⁷GDP cocrystal structure suggested that a conserved hydrophobicsurface feature on the concave dorsal face of eIF-4E is a potential sitefor interaction with eIF-4G and PHAS-I. Residues of eIF-4E that areproposed to participate in this binding include Val-69, Glu-70, Trp-73,Leu-131, Gly-139, Glu-140 and Asp-143 (Marcotrigiano et al., 1997,supra). The finding that substitution with alanine of the amino acidbetween residues 112 to 121 did not disrupt PHAS-I binding is consistentwith the proposed binding site of PHAS-I. In addition, the observationthat mutations in eIF-4E that altered binding to the m⁷G cap structuredid not impair the ability of eIF-4E to bind PHAS-I argues that theoverall structure of the eIF-4E variants was maintained. The m⁷GDPbinding site of eIF-4E is located on the opposite side of the beta sheetfrom the PHAS-I binding site (Marcotrigiano et al. 1997 supra). Thus anychanges in structure due to mutagenesis are probably localized to theside of the beta sheet that has the m⁷G binding site.

The invention is set forth in further detail by description of specificembodiments as set forth in the following examples.

EXAMPLES

Materials—T7 polymerase driven prokaryotic expression vectors andBL21(DE3) strain of E. coli were obtained from Novagen (Madison, Wis.)and are described in detail elsewhere (Studie, S. et al. [1990] Meth.Enzymol. 185:60-89). IPTG was from Fisher Biotech. Hep G2 and 184Amammary carcinoma cells were from the American Type Culture Collection(Bethesda, Md.). Fetal bovine serum was from HyClone (Logan, Utah).m⁷GTP and Protein A Sepharose were purchased from Pharmacia LKBBiotechnology Inc. (Piscataway, N.J.). m⁷GDP was from Sigma Chemical Co.(St. Louis, Mo.). [³²P]Nucleotide triphosphates were from Dupont-NEN and[³⁵S]methionine was from ICN Biomedicals Inc. (Costa Mesa, Calif.). DNAsequencing reagents (Sequenase version 2.0) were from United StatesBiochemical (Cleveland, Ohio). A Coy Model 50 Tempocycler was used forthe PCR studies.

Example 1

Construction of the eIF-4E_(human) expression vector—Standard PCRmethods were used to isolate an eIF-4E_(human) cDNA with engineered 5′Nco I and 3′ Bgl II restriction sites [Sambrook, J. et al. (1989)“Molecular Cloning—A Laboratory Manual” Cold Spring Harbor Press, ColdSpring Harbor, N.Y.; Faloona, F. et al. (1987) Meth. Enzymol.155:335-350]. This permitted subcloning of the eIF-4E cDNA into anexpression vector to produce wild-type eIF-4E. Template cDNAs ofeIF-4E_(human) were derived from both pTCEEC kindly provided by RobertRhoads [Rychlik, W. et al. (1987) Proc. Natl. Acad. Sci. USA 84:945-949]and pCMV-SPORT provided by Deborah Polayes and Joel Jesse (LifeTechnologies, Gaithersburg, Md.). The PCR product was digested with NcoI and Bgl II, isolated using agarose gel electrophoresis and subclonedinto the Nco I and Dam HI sites of pET-3d [Studier, F. W., et al. (1990)Meth. Enzmol. 185:60-89; Sambrook, J. et al. (1989) supra]. Forexpression, the BL21(DE3) strain of E. coli was transformed with thisplasmid. DNA sequence analysis confirmed that the plasmid encodes theoriginal sequence reported for human eIF-4E [Rychlik, W. et al. (1987)supra].

Example 2

Expression and purification of functional recombinanteIF-4E_(human)—Purification was carried out essentially as described byHagedorn, et al. (1997) Protein Expression and Purif. 9:53-60, which isincorporated herein by reference. BL21 (DE3) cells expressing humaneIF-4E were grown in M9ZB media with ampicillin to an OD₆₀₀ of 0.6-0.7at which time cultures were induced with 0.4 mM IPTG [Studier, F. W. etal. (1990) supra]. FIG. 1 shows polyacrylamide gel separation of wholecell lysates at 0, 2 and 3 hours after induction. Culture media wascentrifuged at 500× g for 15 min, and E. coli pellets were suspended in3 ml of 50 mM Tris-HCl pH 8.0, 100 mM KCl, 1 mM EDTA, 1 mM DTT, 1 mMPMSF, and 0.1 mg/ml lysozyme per gram wet weight. Samples were stirredintermittently on ice for 15 min and sonicated. Triton X-100 was addedto a final concentration of 0.1%. The samples were mixed for 15 min at4° C. and centrifuged at 15,000 rpm in a Sorvall SS-34 rotor for 20 min.Supernatants were used as starting material for subsequentchromatography purification steps.

In the first chromatography step we used m⁷GTP Sepharose as described indetail previously [Haas, D. W. et al. (1991) Arch. Biochem. Biophys.284:84-89]. eIF-4E was eluted during m⁷GTP Sepharose chromatography withm⁷GDP. Fractions were analyzed by SDS-PAGE and Coomassie Blue stainingand those containing eIF-4E were concentrated using a Centriprep-10concentrator. The concentrated protein was applied to a Mono Q HR5/5FPLC column in 50 mM HEPES-pH 8.0, 1 mM MgCl₂, and 1 mM DTT at a flowrate of 0.3 ml/min. Following a wash step with the same buffer, proteinswere eluted with a 34 ml linear gradient of 0-500 mM NaCl at 0.3 ml/minin the same buffer. Fractions containing eIF-4E were identified bySDS-PAGE analysis. Results of the purification process are shown inTable 1. FIG. 2 shows polyacrylamide gel analysis of E. coli lysate,affinity-purified eIF-4E. FIG. 3 shows results of an isoelectricfocussing analysis of eIF-4E purified on FPLC.

TABLE I Purification table for recombinant eIF-4E expressed in E. coli.Total Recovery protein Con- Quantity of of soluble Volume centrationeIF-4E eIF-4E (ml) (mg/ml)* (mg)* (%) E. coli culture 1000 — — — Solublelysate 50 10 4 100 m⁷GTP Affinity column 3 0.9 2.5 63 Mono Q FPLC 1 2.12.0 50

* Protein concentrations were estimated using the Bradford method andthe values given are from a representative experiment (Haas et al.[1991] Arch. Biochem. Biophys. 284:84-89).

_ Note: Approximately 1-5% of the eIF-4E expressed in E. coli wassoluble as estimated by Coomassie blue staining of SDS-PAGE analyzedsamples.

Example 3

Fluorescence measurements—Fluorescence measurements were made at 25° C.on a SPEX Fluorolog-T2 spectrofluorometer equipped with a high intensity(450 w) xenon arc lamp. An excitation wave length of 280 nm was used tomonitor the tryptophan fluorescence emission of recombinant eIF-4E at330 nm. Excitation and emission slit widths of 1.4 and 2.0 mmrespectively were used and a 1.0 cm sample cell pathlength was employed.The buffer used for all fluorescence measurements was 20 mM HEPES at pH7.6 and 1 mM DTT (designated Buffer A in FIG. 4). The steady state datawere collected and analyzed as described previously [Carberry, S. E. etal. (1989) Biochemistry 28:8078-8083; Carberry, S. E. et al. (1990)Biochemistry 29:3337-3341. An excitation wavelength of 289 nm was usedto monitor the tryptophan fluorescence emission of the proteins.Excitation and emission slit widths of 1.5 mm and 2.0 mm respectivelywere used and cell path length was 1.0 cm. The results are shown in FIG.4.

Example 4

Immunizations and purification of monospecific rabbit anti-eIF-4Eantibodies—Two female New Zealand white rabbits were immunized by s.c.injections as described in detail elsewhere [Hagedorn, C. H. et al.(1990) FEBS Lett. 264:59-62]. FPLC or SDS-PAGE purified recombinanteIF-4E_(human) was suspended in a 50% emulsion of adjuvant in phosphatebuffered saline and used for immunizations. Between 100 and 400 μg ofprotein were used per animal for each immunization. Boosterimmunizations were given at 4-6 week intervals. FIG. 5 shows results ofimmunoprecipitation of labeled recombinant human eIF-4E using immuneserum of two different rabbits.

Preparation of recombinant eIF-4E and antibody affinitybeads—Recombinant eIF-4E that was purified by FPLC was covalentlycrosslinked to agarose beads (AminoLink, Pierce Chemical Co.) followingthe instructions provided by the manufacturer. Protein A Sepharose(Pharmacia Biotech) was pre-incubated with rabbit pre-immune oranti-eIF-4E serum in lysis buffer (described below) and then washedthree times prior to use in these studies.

Example 5

Metabolic labeling of mammalian cells—Hep G2 and 184A mammary carcinomacells were cultured as described elsewhere [Bu. X. et al. (1993) J.Biol. Chem. 268:4975-4978]. Cells (75 mm² flasks) were incubatedovernight in 3 ml of minimal essential medium without methionine,containing 5% complete medium and 0.3 mCi/ml of[³⁵S]methionine/cysteine. Media was removed, cell monolayers rinsedtwice with phosphate buffered saline and then lysed as described in thenext section.

Isolation of proteins that bind recombinant eIF-4E, anti-eIF-4E antibodyand m⁷GTP affinity beads—Media was removed from labeled cell, rinsedtwice with phosphate buffered saline and then lysed on ice (20 min)using 4 ml of lysis buffer (20 mM HEPES at pH 7.4, 0.5% Triton X-100,100 mM KCl, 2 mM MgCl₂, 50 mM β-glycerolphosphate, 0.5 mM DTT, 1 mMPMSF, 10 μg/ml leupeptin and 10 μg/ml aprotinin). Lysates werecentrifuges for 15 min at 10,000× g (4° C.) and the supernatants removedand divided into four equal aliquots in microfuge tubes. Protein ASepharose antibody beads, recombinant eIF-4E beads, or m⁷GTP Sepharosebeads were mixed with lysates at 4° C. for 45 min. Affinity beads werepelleted by centrifugation for 15 sec in a microfuge and washed threetimes with lysis buffer. Proteins bound to beads were then analyzed bySDS-PAGE and autoradiography [Haas. D. W. et al. (1991) supra]. Theresults are shown in FIG. 6. FIG. 7 shows identification of p220 basedon proteolysis of p220 in poliovirus-infected cells.

Example 6

Modification of eIF-4E binding affinity. A number of individual aminoacids in the region 112-123 was substituted by alanine, using thetechnique of alanine-scanning mutagenesis. Binding constants of thevarious mutants (variants) were measured. Results are shown in Table 2.We expressed and purified milligram quantities of most eIF-4E_(human)variants for detailed analysis using E. coli BL21(DE3)cells transformedwith pET-3d vectors. Cells were cultured, induced with IPTG and lysed inSDS-PAGE sample buffer as described in Example 2. Samples of cells wereanalyzed by SDS-PAGE and Coomassie blue staining. W113A, L117A, Q120Aand Q121A were consistenty expressed at levels 3-4 times more thanwild-type eIF-4E, while L114A expression was lower. The level ofexpression of the other variants was similar to that of wild-typeeIF-4E.

In order to assess the solubility of each variant, cells were lysed andcentrifuged as described, supra. Supernatants prepared from equivalentwet weights of cells were analyzed by SDS-PAGE and Western blotting withanti-eIF-4E antibodies. Supernatants from all variants contained solubleeIF-4E. The lower amount of soluble L114A and the greater amounts ofQ120A and Q121A detected are consistent with their respective lower andhigher levels of expression. W113A and L117A variants, on the otherhand, had high expression levels, but the amount of soluble protein inthe lysate was similar to that of wild-type eIF-4E. Except for the W113Aand L117A variants, no major differences in solubility were observedamong the other alanine variants studied.

Milligram quantities of eIF-4E variants were purified by m⁷GTP-Sepharoseaffinity chromatography and Resource Q FPLC as described for therecombinant wild-type protein [Hagedorn, C. H. et al. (1997) ProteinExpression and Purif. 9:53-60]. Analysis of the variants by SDS-PAGEdemonstrated a high degree purity except for W113A and L117A. Thisproperty of W113A made it impractical to obtain sufficient quantities ofthe variant for further purification. The yield of W113A from a oneliter culture of E. coli was approximately 10 μg, while the yield forwild-type eIF-4E was 102 mg. The yields of purification (mgprotein/liter culture) using m⁷GTP affinity chromatography were also lowfor I115A and L117A (data not shown). These initial results suggestedthat the W113A, I115A and L117A variants had an impaired ability to bindmRNA caps as compared to wild-type eIF-4E. The lower solubility of W113Aand L117A suggest that these variations disrupted the folding of theprotein rather than having a direct effect on cap binding. It isnoteworthy that even though L117A was processed using the samepurification steps as the other variants, it was considerably less pureafter the final step. This was largely due to poor binding in the firstaffinity step.

Circular dichroism analysis: The structural integrity of the variantproteins was assessed by a spectral method, circular dichroism (CD).Structural comparisons of I115A, T116A, N118A and K119A revealed thatthey are all very similar to the wild-type of eIF-4E in both free andm⁷GTP-bound forms. W113A and L117A were not analyzed because ofdifficulties in purifying these variant proteins.

Affinity of eIF-4E variants for the mRNA cap: The binding of eachvariant to the mRNA cap structure was first examined by am⁷GTP-Sepharose binding assay at 4° C. E. coli lysates containingequivalent quantities of eIF-4E protein were incubated with an excess ofm⁷GTP-Sepharose. Proteins that bound to m⁷GTP-Sepharose were analyzed bySDS-PAGE and Coomassie Blue staining. The results demonstrated that theW113A variant barely bound to m⁷GTP-Sepharose, and that I115A and L117Avariants exhibited reduced binding as compared to wild-type eIF-4E. Thisis consistent with the low yields of W113A, I115A and L117A obtainedduring purification of these variants. The binding of all other variantsto m⁷GTP-Sepharose was comparable to that of wild-type eIF-4E.

The preparation of large quantities of recombinant eIF-4E variantsallowed us to directly determine the K_(d) of binding to the mRNA capstructure. Quantitation was performed by measuring the fluorescencequenching of intrinsic tryptophan residues in eIF-4E upon m⁷GTP bindingat 25° C. (Example 3). The K_(d) values of wild-type and variant eIF-4Efor m⁷GTP are shown in Table 1. Except for two variants, these resultswere consistent with those obtained by m⁷GTP-Seepharose binding assays.The exceptions were Q120A and Q121A which had lower affinities for m⁷GTPthan suggested by the m⁷GTP-Sepharose binding assays. This is possibly aconsequence of a lower stability of these variants at the highertemperature in the fluorescence quenching assay.

Ability of eIF-4E variants to bind PHAS-I: The binding of PHAS-I, atranslational repressor protein, to eIF-4E regulates translation andgene expression. To determine if mutagenesis of amino acids in the112-121 region of eIF-4E affected the PHAS-I binding region we examinedthe ability of each variant to bind PHAS-I. Lysates of E. coliexpressing wild-type or eIF-4E variants were mixed with E. coli lysatecontaining recombinant PHAS-I and immunoprecipitated with anti-PHAS-Iantibodies. Samples were analyzed by SDS-PAGE followed by immunoblottingwith anti-eIF-4E antibodies. All eIF-4E variants were able to bindPHAS-I at a level similar to that of wild-type. The amount of PHAS-Iinmmunoprecipitated in these samples was shown to be very similar bystripping and reprobing the nitrocellulose membranes with anti-PHAS-Iantibodies. These results indicate that mutagenesis of the Arg-112 toGln-121 region of eIF-4E did not disrupt its interaction with PHAS-I.

Translational activity of eIF-4E variants: The ability of eIF-4Evariants to initiate translation was examined in rabbit reticulocytelysates which were depleted of endogenous eIF-4E by m⁷GTP-Sepharosechromatography [Svitkin Y. V. et al. (1996) EMBO J. 15:7147-7155].Western blotting analysis using anti-eIF-4E antibodies detected noeIF-4E in lysates after chromatography. The addition of wild-type eIF-4Eto the translation mixture resulted in 2-4 fold increase in thetranslation of globin mRNA. This increase in translation wascap-dependent since addition of m⁷GDP inhibited the eIF-4E-dependenttranslation. The relative abilities of eIF-4E variants to restore globinmRNA translation was tested for all variants except W113A. Except forL117A, all variants were able to initiate translation to a level similarto that of wild-type eIF-4E.

Cell-free translations: Nuclease-treated rabbit reticulocyte lysates(Promega) were depleted of endogenous eIF-4E by chromatography onm⁷GTP-Sepharose according to the previously described protocol (Svitkinet al. 1996, supra). Aliquots of eIF-4E-depleted lysates were stored inliquid nitrogen vapor. eIF-4E depletion was verified by Western blottinganalysis (data not shown). Cell-free translations were performed at 30°C. as described in detail in the Promega technical manual. Reactionmixtures contained 75 ng wild-type or variant eIF-4E (FPLC purified), 80ng/ml globin mRNA, 10 μCi [³⁵S]methionine (>1000 Ci/mmol, ICN) and 10 μlreticulocyte lysate in a final volume of 16 μl. In the case of L117A,which was not highly purified, a sufficient quantity of the preparationwas added to approximate 75 ng of L117A. As a control for cap-dependenttranslation, 1 mM m⁷GTP was added to a complete incubation containingwild-type eIF-4E. Following 60 min of incubation at 30° C., 7 μl of thereaction mixture were spotted on a 1 cm² Whatman 3 MM filter paper andair dried for 10 min. Filters were soaked in 5% trichloroacetic acid(TCA) with 1 mM methionine for 10 min on ice, rinsed with fresh 5% TCAand then boiled for 10 min in 5% TCA. Following another wash with 5%TCA, filters were rinsed with 95% ethanol and then with acetone. Driedfilter papers were placed in vials with scintillation liquid and[³⁵S]methionine incorporated into protein was quantitated by liquidscintillation spectrometry.

TABLE 2 Binding constant Wild type Amino acid replacement K_(d) (82 M)amino acid Wild type 0.14 R112 → A 0.11 Positively charged W113 → A Verylow binding Hydrophobic L114 → A 0.11 Hydrophobic I115 → A 0.31Hydrophobic T116 → A 0.14 Polar, uncharged L117 → A 0.20 HydrophobicN118 → A 0.07 Polar, uncharged K119 → A 0.04 Positively charged Q120 → A0.22 Polar, uncharged Q121 → A 0.37 Polar, uncharged

Further modifications and variations can be made according to theprinciples and teachings disclosed herein, including, but not limitedto, improvements in yield of soluble eIF-4E from lysate, other kinds ofamino acid substituion, both as to locus and identify of substituentamino acid, and improvements and optimization of RNA transfection usingeIF-4E-m⁷G-RNA or variant eIF-4E-m⁷G-RNA.

We claim:
 1. A purified human variant eIF-4E protein having greaterbinding affinity for m⁷G-RNA than natural human eIF-4E, said proteinhaving an amino acid substitution of alanine for asparagine at aminoacid position number
 118. 2. A human variant eIF-4E protein in purifiedform, said protein having greater binding affinity for m⁷G-RNA thannatural human eIF-4E, and said protein having an amino acid substitutionof alanine for lysine at amino acid position number
 119. 3. A purifiedhuman variant eIF-4E protein having an amino acid substitution in theregion of amino acids 112 and 114-121, said protein having alteredbinding affinity for m⁷G-RNA compared to natural human eIF-4E.
 4. Theprotein of claim 3 having an amino acid substitution of alanine forisoleucine at amino acid position number
 115. 5. The protein of claim 3having an amino acid substitution of an alanine for glutamine at aminoacid position number
 121. 6. The protein of claim 3 having greaterbinding affinity for m⁷G-RNA than natural human eIF-4E protein.